The goal of this proposal is to use protein engineering to develop inhibitors of a protease thought to play a central role in the invasion and metastasis of cancerous cells. The macromolecular activity against the cancer-associated serine protease urokinase-type plasminogen activator (UPA). Understanding the basis of the specificity and potency of macromolecular UPA inhibitors will aid in the design of small polypeptide inhibitors which emulate the macromolecular activity. Preliminary results haveshown that the ecotin mutant m84R/M95R has a 2800-fold greater binding affinity for UPA compared with native ecotin. Computer homology and molecular modeling performed at the CGL would assist in the identification of ecotin residues that may be mutated for greater inhibition and specificity towards UPA. These identified residues can be modified by site-directed mutagenesis and mutants will be assayed by standard enzymatic techniques. Further, computer modeling would assist the identification of small regions of ecotin that appear to have significant binding interactions with UPA. These regions will be randomized by multiple site saturation mutagenesis or by DNA shuffling, and phase display will be used to express these libraries. Understanding the mechanism by which macromolecular inhibitors can strongly inhibit proteases will aid in the development of small peptide and non-peptide inhibitors. These small molecule inhibitors can be designed to emulate the interactions of the macromolecular inhibitor and will be developed for the use in protease inhibitor cocktails that block primary tumor growth.